Chemotherapeutic Anti-proliferative Drugs
Anti-proliferative drugs, also known as anti-metabolites, act by inhibiting crucial metabolic processes, and are commonly used in the treatment of diseases involving abnormal cell proliferation, such as tumors. However, the utility of these drugs is severely hampered by their excessive toxicity and adverse side effects on healthy cells of the treated patient. Therefore, it would be advantageous to be able to reduce these adverse effects by the use of a prodrug having decreased toxicity.
The use of prodrugs to impart desired characteristics such as increased bioavailability or increased site-specificity of known drugs is a recognized concept in the state of the art of pharmaceutical development. The use of various blocking groups, which must be removed in order to release the active drug is also known in the background art. Commonly, one or more blocking groups may be attached via an available amine, hydroxyl group or other functional reactive group on the drug to yield an amide or an ester. This type of prodrug may be cleaved by non-specific esterases to release the active principle in a sustained-release fashion over a prolonged period of time compared to the native drug species.
Methotrexate (MTX), for example, is an effective anti-proliferative drug commonly used in cancer therapy. It is an analogue of dihydrofolate that inhibits the enzyme dihydrofolate reductase (DHFR), thus depleting intracellular tetrahydrofolate (FH4), which is an essential co-factor required for the de novo synthesis of purine nucleotides.
MTX, Mephalan and Chlorambucil are valuable drugs in the treatment of many rapidly growing tumors, however, their use is limited by the frequency and severity of side effects. Unwanted side effects include toxicity to all rapidly dividing normal cells, such as stem cells in the bone marrow, epithelial cells of the intestinal tract, hair follicle cells, etc.
Another major problem in chemotherapy, which is particularly relevant in the case of anti-metabolites, is inherent or acquired resistance of tumors to cytotoxic drugs. For example, development of resistance to MTX frequently follows prolonged exposure to this drug. Resistance may be due to new mutations induced by the clinical treatment, or to positive selection, by the treatment regimen, of pre-existing resistant mutant cell. Known mechanisms for development of resistance involve impaired transport of MTX into cells, e.g. by mutations in the Reduced Folate Carrier (RFC), over expression of the target enzyme DHFR, or mutations in the enzyme responsible for polyglutamination of reduced folates (FPGS).
A more severe problem in the clinic is the phenomenon of multi-drug resistance (MDR), which is a resistance to a broad spectrum of structurally unrelated cytotoxic drugs. MDR is mediated by transmembrane “pumps”, which actively expel chemotherapeutic drugs from the tumor cells. MDR significantly limits the efficacy of many cancer chemotherapy regimens and is a major factor in the failure of cancer chemotherapy.
It would, therefore, be most advantageous to have drug derivatives that are specifically targeted to or selectively active in the diseased cells rather than in the healthy cells, thus reducing undesirable side effects. It would also be desirable to generate new anti-proliferative agents that overcome drug-resistance, as well as agents that are active as cytotoxic drugs but lack or have a reduced ability to provoke MDR phenotype.
For specific cytotoxic drugs it has been suggested that the therapeutic index of such drugs might be increased if the drug is covalently bound to a peptide that would be cleaved in the vicinity of the tumor cells by the action of certain proteases. This approach has been suggested for peptide conjugated Methotrexate (Kuefner et al., 1989) and for Arabinofuranosyl cytosine (ara-C) lipid-peptide-drug conjugates (Menger et al., 1994).
Glycosaminoglycans Binding Proteins
Many different types of cell surface polypeptides or glycoproteins have been utilized for targeting drugs to malignant cells, with various degrees of success.
The use of specific cell surface complex sugars as cell surface markers is much less well developed. In part this is due to the fact that the expression of these structures cannot be followed in terms of gene transcription. In other words, the complex sugars are the product of varying expression of the glycosylation enzymes, and cannot be traced directly as gene products.
Proteoglycans are composed of long, unbranched sugar polymers, called glycosaminoglycans (GAGs), which are covalently linked to a core protein. The proteoglycans constitute the extracellular matrix, such as the cartilage, the basement membranes, and the connective tissue. They are also found on the cell surface (Bernfield, M. et al. 1992). Virtually all epithelia express cell-surface proteoglycans, represented principally by glypicans and syndecans. Glypicans are glycosyl phosphatidyl inositol (GPI)-linked molecules, and bear glycosaminoglycans exclusively of the heparan sulfate type. Syndecans are transmembrane proteins, and are decorated with chondroitin sulfate and with heparan sulfate polymers. Syndecans exhibit a complex pattern of cell and development specific expression, however, the molecular mechanisms responsible for this expression have not been fully explored. It was shown, for example that during wound healing the expression of syndecan-1 and -4 is induced. In the case of glypicans, it was shown that glypican-1 is strongly expressed in human pancreatic cancer, whereas its expression is low in normal pancreas.
A variety of regulatory proteins bind tightly to GAGs, including growth factors, cytokines, chemokines, extracellular matrix proteins, cell adhesion molecules, lipid binding proteins, enzymes, and blood coagulation factors. The role of heparan sulphate proteoglycans (HSPGs) in growth factor signaling has been best characterized with respect to fibroblast growth factors (FGFs), which require the presence of heparan sulfate for high affinity binding to their tyrosine kinase receptors (Yayon, A., et al. 1991). Several other growth factors have been found to exhibit a strong requirement for a HSPG co-receptor in their signaling. These include heparin binding EGF-like growth factor (HB-EGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) (Yamada, Y. et al., 1997), PDGF, TGF-beta, and other types of growth factors.
Vascular endothelial growth factors (VEGFs) are mitogens for endothelial cells and are potent angiogenic factors in vivo. VEGF-165 contains the peptide encoded by exon-7 of the VEGF gene, confers on VEGF-165 the ability to bind heparan-sulfate molecules. VEGF-145 contains the peptide encoded by exon-6a of the VEGF gene, enabling VEGF-145 to bind ECM (Poltorak et al., 1997).
Several VEGF tyrosine-kinase receptor types have been characterized, these receptors mediates the mitogenic activity and induced cell migration of VEGF. Other VEGF receptors, neuropilin-1 and neuropilin-2 (Gitay-Goren, H., et al., 1992) bind only to the GAG binding forms of VEGF (VEGF-165, VEGF-145) that have GAG binding peptides (axons 6a or 7) of the VEGF gene. These receptors are highly expressed in cancer cells such as human melanoma and carcinoma, but not expressed in normal melanocytes.
VEGFs play a critical role in the process of tumor angiogenesis. This process is essential for tumor progression and for the subsequent process of tumor metastasis.
VEGF soluble receptors have been suggested as an inhibitor of endothelial cell induced proliferation and angiogenesis (Kendall et al. U.S. Pat. No. 5,712,380).
Among the chemokines that are known to bind to heparin the better characterized are Platelet factor 4 (PF4) (Morgan et al., 1977). PF4 is an anti-angiogenic factor that belongs to the CXC Chemokines family. PF4 binds to several receptors that belong to the CXC receptor (CXCR) family involved in angiogenesis. Kaposi's sarcoma cancer is indicated by uncontrolled angiogenesis that is associated with KSHV (Kaposi's sarcoma associated herpes virus) that produces the CXCR-2 receptor homolog.
Injection of fluorescent PF4 to hamsters showed concentration of PF4 at capillary endothelial cells at sites of active angiogenesis. PF4 is accumulated at high concentrations in extra cellular matrix and basement membrane due to its GAG binding ability.
PF4 can bind cell surface proteoglycans, and can be accumulated in the intracellular compartments (Neufeld at al., personal communication). Peptide from its GAG binding domain inhibited melanoma tumor growth in mice xenograft, though it had no effect on cancer cells in-vitro. CXC chemokines have been suggested as therapeutic molecules in modulating the angiogenic and angiostatic responses (U.S. Pat. No. 5,871,723).
Proteolytic Enzymes and Cancer Cells
Cancer invasion involves a proteolytic degradation of extracellular matrix in the surrounding normal tissue. Excess matrix degradation is one of the hallmarks of cancer, and is an important component of the process of tumor progression (Fidler, I. J., 1997). In order for invasion and metastasis to occur, the tumor cell must bypass the basement membrane by degrading the components of the ECM.
Various proteases, in particular the serine protease plasmin, and a variety of matrix metalloproteinases (MMPs), have been implicated in tumor invasion. Plasmin is formed from the inactive zymogen plasminogen by the plasminogen-activators. Plasminogen is produced in the liver and is present extracellulalrly throughout the body. One of the plasminogen-activators, the urokinase plasminogen activator (uPA), is synthesized as a pro-uPA that binds with high affinity to a cell-surface-bound receptor, the uPA receptor (uPAR). Receptor binding of pro-uPA strongly enhances the overall reaction leading to plasmin formation (Dano, K. et al., 1994). Clinical findings have shown that there are elevated tumor antigen levels of Plasminogen Activator (uPA, tPA) and its receptor uPAR in cancer cells and tumors and it plays a role in tumor invasion and metastasis (Koopman et al., 1998; Schmidt et al., 1997).
The MMPs comprise of a large family of over 20 proteins that can degrade all the known components of the extracellular matrix (Massova, I. et al. 1998). MMPs were identified in the tissues surrounding invasive cancers, and show over expression in malignant tissues.
The human aspartic proteinases include cathepsin D, cathepsin E, pepsinogen A, pepsinogen C, and rennin (Taggart, R. T., 1992). Cathepsins D and E are significantly elevated in various cancers and metastases, hence applied as tumor cell markers of epithelial cancers (Matsue, K. et al., 1996)
Nowhere in the background art is it taught or suggested that it is possible to use peptides as drug carriers useful to target prodrugs to tumors.